Inflammasome-Mediated Interleukin-1β Drives Neutrophilic Airway Inflammation in Cystic Fibrosis Open Access

Rao, Sanjana K. (2016)

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Cystic fibrosis (CF) patients experience progressive airway destruction due to opportunistic pathogen colonization and neutrophil-dominated chronic inflammation. In order to investigate the inflammatory response promoting disease progression, we assessed neutrophil inflammasomes and the resulting activation of the IL-1 (interleukin-1) pathway, consisting of pro-inflammatory IL-1β signaling and an endogenous inhibitor, interleukin-1 receptor antagonist (IL-1RA). Specifically, we performed ex vivo assays of CF human blood and airway samples with comparison to healthy control (HC) subjects and in vitro neutrophil inflammasome stimulations. Further, we assessed bioactivity of IL-1β to identify the potential role of IL-1RA in modulating the IL-1β activatory cascade, and to investigate potential correlations with disease severity. Results show that IL-1β production is significantly elevated in CF, compared to HC and chronic obstructive pulmonary disease (COPD) controls, confirming that CF disease is linked with IL-1 signaling. Furthermore, we observed a strong correlation between neutrophil numbers and IL-1β levels, consistent with a pro-inflammatory loop of neutrophil recruitment and IL-1β secretion that itself recruits more neutrophils. Results from inflammasome stimulation and follow-up in vitro assays together demonstrate that IL-1β secretion may not be concomitant with cell death, contrary to conventional belief. Finally, using a cell line genetically engineered to detect bioactive IL-1β, we found that IL-1β in CF airway samples is indeed bioactive and that this bioactivity is closely regulated by IL-1RA. Ultimately, results gathered strongly suggest that IL-1β is a clinically relevant modulator of CF, and a potential target for therapeutic intervention.

Table of Contents

I. Introduction. 1

i. Background. 1

ii. Genetic basis of CF. 1

iii. Pathophysiology of CF airway disease. 2

iv. PMN-mediated inflammation. 2

v. Inflammasome activation. 3

vi. Modulation of IL-1β pathway. 4

vii. Study rationale. 5

II. Materials and Methods. 5

i. Human subjects. 5

ii. Sample collection processing. 6

iii. ELISA. 6

iv. Human Embryonic Kidney (HEK)-Blue Cells. 8

v. in vitro inflammasome activation. 10

vi. LDH Assay. 11

vii. Statistical analysis. 12

III. Results. 12

IV. Discussion. 14

V. Tables and Figures. 18

i. T1. 18

ii. F1. 18

iii. F2. 19

iv. F3. 20

v. F4. 21

vi. F5. 22

vii. F6. 23

viii. F7. 24

ix. F8. 25

x. F9. 26

xi. F10. 27

xii. F11. 28

xiii. F12. 29

xiv. F13. 31

VI. References. 32

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